The discovery of the coreceptors for HIV- 1, HIV-2, and SIV five years ago initiated an era of rapid progress that elucidated mechanisms responsible for virus entry and tropism, identified genetic factors that impact virus transmission and disease progression, and led to the identification of new drug and vaccine targets. While the expression patterns of the major HIV coreceptors, CCRS and CXCR4, coupled with the differential use of these receptors by HIV-1 strains explains much about viral tropism, it is becoming increasingly clear that identifying what receptors are used by a virus strain is but the first step. How a receptor is used to effect entry likely have major implications for virus tropism, pathogenesis, and sensitivity to entry inhibitors. To explore these relationships, our understanding of how Env engages coreceptors has to be improved. While the V3 loop and V2 regions are largely responsible for coreceptor choice, a region in the gp120 "bridging sheet" that is remarkably well conserved has been shown to play a role in CCRS binding. Whether this domain represents a binding site for other coreceptors remains to be determined. Studies in this area are the subject of Specific Aim #1, and will reveal regions in Env that are important for binding affinity, and triggering the conformational changes that lead to membrane fusion. In Specific Aim #2, we will explore the biological consequences of differences in Env-coreceptor interactions. Our studies have shown that some viruses bind their coreceptors with high affinity, while others bind with low affinity. Given that several receptor binding events are required to form a fusion pore, we hypothesize that viruses with high affinity will fuse more rapidly and be better able to infect primary cells that express low levels of coreceptor. In Specific Aim #3, we will study the effects of differences in Env-coreceptor interactions on sensitivity to different classes of entry inhibitors. We have evidence that enhanced receptor affinity will result in increased resistance to entry inhibitors such as T20, which targets structural intermediates of the fusion process. Finally, we have derived a novel HIV-2 variant that exhibits high affinity for CXCR4, utilizes both CXCR4 and CCRS in the absence of CD4, and remains replication competent even when V1N2 and 60 percent of its V3 loop are deleted. The high affinity for CXCR4 displayed by this isolate is particularly unique and affords an opportunity to map and manipulate the coreceptor binding site for structural and biological studies in several novel ways as described in Specific Aim #4.